The ability to cryopreserve mammalian oocytes with high success rates in an easily reproducible manner has not yet been achieved. Cryopreservation of mouse oocytes, for example, could be valuable for the long term storage of genetically important strains of mice and would serve as a model for oocyte cryopreservation in humans, commercial livestock, and endangered species. This technology could also help alleviate the legal, ethical, and religious problems associated with the storage of human embryos, as well as improve current embryo freezing protocols. Although offspring have been produced from frozen-thawed oocytes in the mouse, human, bovine, and rabbit (Whittingham, 1977; Al-Hasani et al., 1987; Fuku et al., 1992; Chen 1986; Van Uem et al., 1987), results have been variable and not sufficiently successful to make oocyte cryopreservation routine.
In addition to oocyte freezing, cryopreservation and transplantation of ovarian autografts have been somewhat successful in mice and sheep (Gosden et al., 1994; Gunasena et al., 1997). The ovarian tissues in these reports were frozen using simple embryo freezing protocols. Ovarian tissue survived freezing and was able to continue development after transplantation to the reproductive tract or kidney capsule, yielding growing follicles.
Current cryopreservation protocols have evolved from embryo freezing methods that produced offspring in mice, cows, and sheep (Whittingham et al., 1972; Willadsen et al., 1976, 1978). Cryopreservation procedures for mouse and other mammalian embryos are now relatively efficient, but these techniques cannot be used reliably for oocyte freezing. Studies cryopreserving mouse oocytes report very different survival and fertilization rates (Carroll et al., 1993; Carroll et al., 1990; Cohen et al., 1988; George et al., 1994; Glenister et al., 1990; Gook et al., 1993; Whittingham et al., 1977). The variability in the success of mouse oocyte freezing is most likely due to modifications in freezing protocol and/or the use of different cryoprotectants. Although these protocols differ, they rely on the same basic cryobiological principals.
A particularly important area that would benefit greatly from advances in cryopreservation technology is assisted human reproduction. In this field, one or both partners may have a fertility problem. In order to overcome these problems, eggs are harvested from the mother, and sperm from the father. The sperm are then used to fertilize the eggs, and one or more developing embryos are then replaced to the uterus of the mother. Typically, egg maturation is induced pharmaceutically prior to harvesting, and the sperm must be available to fertilize the eggs. Generally, only a very limited number of surgical harvesting procedures may be conducted on an individual, and the number of eggs replaced in the mother is limited in order to avoid multiple live births. Therefore, there has been a need to preserve harvested eggs, sperm, fertilized eggs, and embryos.
The ability to successfully and reproducibly cryopreserve oocytes, which is not yet possible, would allow women to have their eggs frozen until a time when they have found a suitable sperm donor (possibly a future husband) and then thaw their frozen eggs and fertilize them. The resulting embryos can be replaced into the patients' uterus, after it has been prepared to receive the embryo using hormones and known techniques, to allow for implantation, fetal development, and ultimately birth. Ethically, there are issues involved in preserving fertilized eggs or embryos, that are more significant than those associated with freezing unfertilized germ cells. Cryopreservation of oocytes would avoid the ethical concerns surrounding embryo freezing in humans and offer another option to couples with infertility problems. Storing oocytes early in life, such as when the mother is in her 20's and early 30's, when healthy eggs tend to be produced, and therefore when they are more apt to be fertilized and result in viable offspring, would greatly improve the chance of a pregnancy later in life, rather than the relatively poor pregnancy rates produced when participating in an in vitro fertilization (IVF) program at 40 years old or older.
Cryopreservation of oocytes, especially from humans, in a reproducible and efficient manner has been generally unsuccessful according to known techniques. It is noted, however, that offspring have been produced from frozen oocytes of several species, including humans. An improved cryopreservation medium would benefit oocyte storage and may also provide a more successful way of freezing embryos, thereby improving the possibilities for pregnancy.
Cryopreservation technology is applicable to other species besides humans. In commercial livestock, improvements in oocyte or embryo cryopreservation could greatly improve genetic management of populations and the number of offspring generated, resulting in a significant time savings and efficiency. In endangered animals, any improvement in embryo freezing or the development of a method to freeze eggs could lead to an increase in the number of offspring produced, enhancement of the genetic quality of the offspring, improvement of the population's genetic health, elimination of both the cost and risk of transporting live animals for reproductive purposes, and possibly even a delay or prevention from extinction of certain species.
Cryopreservation of oocytes from commercial livestock would provide a valuable means of saving and distributing important genetic material. Oocytes from animals exhibiting commercially valuable traits such as increased milk production could be stored.
Cryopreservation of oocytes from endangered species would provide an invaluable method of salvaging important genetic material. Provided that the eggs could be thawed, fertilized, and produce fertile offspring, and given that sperm is relatively easy to store, species could be stored indefinitely, virtually eliminating the risk of extinction. Cryopreserved oocytes could be easily transported globally, providing a source of genetic information to better aid in managing populations. In this manner, underrepresented genes from founder animals could be reintroduced into the population at any time, even 200 years from now. Because frozen oocytes (and sperm) can be distributed easily and cost effectively, the possibilities of improving genetic diversity and the overall health of a population are intriguing. Oocytes collected in the field can be frozen and infused into the captive population to improve its genetic health. With this technology in place frozen zoos can become a reality.
Biological cryopreservation systems are well known. They allow cells to be frozen, for example at -20.degree. C. or below, for extended periods, and resume normal cell activity after thawing. Typically, problems encountered include intracellular ice formation (IIF) and osmotic imbalances that result in cellular disruption. It is the prevention of IIF that many prior methods are directed.
Cryopreservation of cells involves dehydration, introduction of a cryoprotectant, and cooling to a low temperature, usually from -30.degree. C. to -80.degree. C., before plunging in LN.sub.2. The first objective is the removal of water from the cell, which when cooled below its melting point forms ice crystals that can damage intracellular organelles as well as the cell membrane (Mazur, 1977). The osmolality of the extracellular solution increases as the outside water freezes, causing the water to passively exit the cell. More ice forms at lower temperatures resulting in continued cellular dehydration. The next objective for freezing cells concerns the combining of any residual water left in the cell with a cryoprotectant, in order to form a glass-like structure when solidified, thereby preventing IIF. Because the melting point of water is reached both during freezing and thawing, IIF can occur at either time. Damage may therefore occur when the cell is exposed to elevated concentrations of electrolytes and/or IIF. IIF can be catalyzed by the presence of extracellular ice surrounding the cells (seeding) or heterogeneously by intracellular structures. In the presence of cryoprotectants however, IIF resulting from either seeding and/or heterogeneous ice nucleation does not occur (Toner et al., 1991), suggesting that IIF may not be a problem when freezing oocytes in the presence of a cryoprotectant. Therefore, electrolytes including sodium would appear to impart the majority of the damage during cryopreservation. Cellular disruption by sodium ions could alter the cell membrane and/or intracellular organelles. Lovelock (1954) hypothesized that cellular damage is caused by an increase in electrolyte concentration, causing destabilization of membranes. Mazur et al. (1974) further demonstrated that the cell surface is a major site of freezing damage. In his study, Mazur showed that the nonpermeating solute sucrose was as effective in protecting erythrocytes from freeze/thaw damage as the permeable cryoprotectant glycerol. The majority of damage to mouse oocytes during freezing may be caused by sodium ions, but we cannot rule out the possibility of IIF. Whether cellular demise is caused by exposure to elevated sodium ion concentrations during the freeze or thaw, or a problem with the transport of sodium ions across cell membranes remains to be elucidated. Sodium ions have a radius of about 0.95 .ANG., while lithium has a radius of 0.60 .ANG. and potassium has a radius of about 1.33 .ANG.. The majority of past cryopreservation studies that have focused on IIF, cryoprotectants, and altering freezing protocols have been unable to significantly improve oocyte freezing. Therefore, the type of cryoprotectant used, or the freezing protocol may already be adequate for oocyte freezing and IIF may not be the major problem presumed by the prior art.
Standard embryo cryopreservation techniques are remarkably similar. In general, embryos are exposed to a cryoprotectant (dimethyl sulfoxide (DMSO), 1,2-propanediol (PrOH), glycerol, ethylene glycol), diluted in a simple sodium-based salt solution for 5-15 min to allow uptake of the cryoprotectant. The embryos are then cooled quickly (-2.degree. C./min) to about 7.degree. C. at which point they are seeded, cooled slowly (-0.3.degree. C. to -0.5.degree. C./min) to about -30.degree. C. or below, and then plunged directly into liquid nitrogen (LN.sub.2). Embryos can also be rapidly frozen or vitrified, but only using very elevated cryopreservative concentrations (2M to 6M) that are toxic to cells when they are exposed for more than a few minutes. Following cryopreservation the embryos are thawed and cultured. Thawing procedures differ, but very little. Two basic concepts are involved in the thawing process, 1) removal of cryoprotectant and 2) rehydration of the embryo at a rate so as not to rupture the cell membrane, usually with the use of sucrose. These freezing and thawing procedures work relatively well for embryos, but do not allow the successful storage of oocytes. The exact reason(s) why embryos can survive and oocytes cannot are unknown, but membrane damage due to IIF, ion loading, and/or osmotic stress are suspect. Researchers have mainly focused on the problem of IIF and osmotic stress by modifying freezing procedures (slow vs. fast), thawing procedures (slow vs. fast), and methods for the removal of the cryoprotectants with little attention to the cryopreservation media formulation, which is presumed to be optimal.
Researchers have tried unsuccessfully to cryopreserve oocytes in a reproducible manner using one or more cryoprotectants. In general, PrOH and DMSO are the most common cryoprotectants used today for the cryopreservation of oocytes and embryos. Although all of the pregnancies in the human have resulted from oocytes frozen in DMSO, PrOH is the cryoprotectant of choice because of its greater permeability, reduced toxicity, and improved success in storing human embryos (Gook et al., 1995; Imoedemhe and Sigue, 1992; Lassalle et al., 1985; Mandelbaum et al., 1987; Testart et al., 1986). Mouse oocytes have been frozen using PrOH, but with poor overall results (Gook et al., 1993; Todorow et al., 1989). Todorow et al. (1989) reported survival and fertilization rates of 63% and 27%, respectively. When PrOH was used in combination with DMSO, survival and fertilization rates increased to 87% and 42%, respectively. Numerous studies have reported survival and fertilization rates of up to 79% and 50%, respectively, for mouse oocytes cryopreserved using DMSO (Glenister et al., 1987; George and Johnson, 1993; Carroll et al., 1989; Todorow et al., 1989; Schroeder et al., 1990; Aigner et al., 1992; Bouquet et al., 1992). Although one laboratory has reported survival rates of up to 91%, fertilization up to 78%, and blastocyst formation as high as 54.4% (Carroll et al., 1993), it remains to be seen whether their results can be easily reproduced.